Microbial community structure and biogenic amines content variations in chilled chicken during storage

Abstract The aim of this study was to investigate the sensory indicators, biogenic amine contents, and bacterial community structure and diversity of chilled chicken stored at 4°C under aerobic conditions. Bacterial diversity and dominant bacteria were analyzed using high‐throughput sequencing technique (HTS). The relationship between biogenic amine contents and microbial community structure was studied. The results showed that contents of putrescine and cadaverine increased significantly with storage time. Proteobacteria was absolutely dominant flora at the phylum level. The predominant spoilage bacteria found in chicken thighs were Pseudomonas, Acinetobacter, Aeromonas, Shewanella, and Yersinia, and the difference with chicken breasts was related to the presence of Myroides and absence of Yersinia. Myroides, Yersinia, and Shewanella were reported for the first time as an important contributor to the spoilage‐related microflora. Bacterial diversity and richness indices showed fluctuating and decreasing trend with storage time. The redundancy analysis showed that the relative abundance of Pseudomonas, Yersinia, and Janthinobacterium was positively related to the contents of putrescine, cadaverine, and tyramine, while Shewanella and Aeromonas showed positive relationship with putrescine content. Furthermore, positive relationship of Myroides and Desulfovibrio with the contents of cadaverine and tyramine was proposed for the first time. The key findings of this study can provide experimental data for food safety monitoring during refrigerated storage and preservation for poultry meat products.

Chicken could be contaminated by a variety of microorganisms during storage process. At present, the research on microbial community structure and diversity during the storage of meat products was conducted by various approaches. Among them were microbial culture methods combined with 16S rDNA sequencing technology (Zhang et al., 2015;Olsson et al., 2003), microbial culture methods combined with identity of fatty acid profiles of the isolates (Hinton et al., 2004), and denaturing gradient gel electrophoresis (DGGE; Liang et al., 2012;Jiang et al., 2011;Dias et al., 2013;Bekaert et al., 2015;Bassey et al., 2021). However, to the extent of our knowledge, HTS has not been studied yet or the microbial community structure and diversity during the storage of meat products. The traditional methods of microbial culture in which use of selective medium for specific microorganism growth is laborious have many limitations (Mohammed et al., 2020). This could be due to complex microbial composition in meat, and only 1% of the total microbial stains found in nature can be cultured on selective medium (Giraffa & Neviani, 2001). DGGE technology could not accurately reflect the microbial community structure and diversity during the storage of meat products.
HTS can sequence hundreds of thousands to millions of DNA molecules in one time, and targeted resequencing is a great application in HTS, so its read is shorter than previous sequencing technique. HTS is characterized by perfect quantitative function, ability to detect DNA species, and can test the abundance of species in samples (Qi et al., 2021). Furthermore, HTS can highlight predominant microorganisms from the phylum, class, order, family, genus, and species levels, thus reflecting deeper analysis of microbial community structure and diversity . HTS has been used generally to study the communities of the environment and human on the ground (Chen et al., 2020), but only a few studies have used this technique to analyze microbial communities of chilled poultry meat.
This work deals with the issues of chilled poultry meat, such as short shelf life, low meat quality, and food safety by investigating the sensory indicators, biogenic amine contents, and bacterial community structure and diversity. Microbial diversity and dominance of specific strains were analyzed by HTS. The relationship between biogenic amine contents and microbial community structure was examined. Some deterioration phenomena like mucous, moldy, foaming, and strong rotten sour odor were investigated in chilled chicken.
The microbial mechanism that affects chilled chicken putrefaction was explained.
1 | MATERIAL S AND ME THODS

| Materials
Chilled chicken thighs and breasts were obtained from Furun Co., Ltd. slaughtering and processing unit. About 10 kg chicken thighs and 15 kg chicken breasts were divided into 10 sterile plastic sampling bags and labeled as CL0, CL3, CL6, CL8, and CL10 for chicken legs and CB0, CB3, CB6, CB8, and CB10 for chicken breasts. The samples were transported immediately to the laboratory in enclosed polystyrene type of packaging unit having ice blocks (4°C) under aerobic environment. The CL0 and CB0 were chosen to explore the microbial flora, structure, and diversity at the same day, while other samples were stored at 4°C under aerobic environment and analyzed for the microbial diversity after 3rd, 6th, 8th, and 10th days, respectively. To minimize the error each treatment was repeated 3 times.

| Sensory analysis and evaluation
Sensory assessment of all the collected samples was performed by seven experts who have rich experience in chicken meat evaluation. Color, texture, appearance, and odor of chicken breast and thigh samples were evaluated. Sensory score criteria of chilled chicken during storage was shown in Table 1. A three-class evaluation scheme was used: Class 1, fresh meat; Class 2, secondary fresh meat; and Class 3, spoiled meat.

| Bioinformatic analysis
After sequencing, initial DNA fragments were congregated by using FLASH (Magoc & Salzberg, 2011). Quantitative insights into microbial ecology (QIIME; Caporaso et al., 2010) and UPARSE pipeline (Edgar, 2013) software packages were employed to analyze each sequence reads assigned to every sample by their barcode. At first, QIIME quality filters were used to filter the sequence reads and then clustered into operational taxonomic units (OTUs) at a 97% identity threshold using UPARSE pipeline. The commissary sequence of every OTU was chosen and designated to taxonomic data by utilizing RDP classifier (Wang et al., 2007).
Alpha diversity analysis included witnessed species, Ace and Chao1 estimators, Simpson and Shannon diversity indices, and Good's estimate of coverage. The term "Simpson's diversity index" reflected to Simpson's index (D), Simpson's index of diversity (1-D), and Simpson's reciprocal index (1/D). This article used the Simpson's index of diversity (1-D), which denotes the likelihood that two individuals haphazardly chosen from a sample will belong to various species.

| Statistical analysis
Whole experiment was repeated 3 times for every date of storage.
Based on the results of all sample species annotations, the top 10 phyla and the top 10 genera were examined (the relative abundance over 1%). A principal coordinate analysis (PCoA) was performed on the weighted UniFrac distance and unweighted pair group method with arithmetic mean (UPGMA) and shown by QIIME software (version 1.7.0).
In addition, correlations between key bacterial community composition and biogenic amine contents were studied by t-value examination obtained from redundancy analysis (RDA) and conducted using Canoco 4.5 (Biometrics). The analyses data were acquired by Microsoft EXCEL (2016) spread sheets.

| Sensory analysis and evaluation of chilled chicken during storage
Sensory analysis and evaluation of chilled chicken during storage at 4°C under aerobic conditions was shown in Table 2. It can be seen that chilled chicken stored for 0 day was fresh meat, at third day was secondary fresh meat, and after sixth day of storage it was spoiled.

| Biogenic amine contents of chilled chicken during storage
The types and contents of biogenic amines detected in chicken thighs and breasts during storage at 4°C under aerobic conditions are described in Table 3. The biogenic amines, tryptamine, phenylethylamine, and histamine, were not detected in chicken thighs and breasts during the whole storage period. The tyramine was not yielded at the initial stage of storage, however, the tyramine contents increased significantly in chicken thighs after sixth day and in breasts after eighth day of storage. Furthermore, the concentrations of putrescine and cadaverine also increased significantly in chicken thighs after sixth day and in breasts after eighth day of storage. As compared with the freshly slaughtered chicken, putrescine concentrations in chicken thighs after 3rd, 6th, 8th, and

| PCoA and UPGMA clustering
Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of microbial communities showed in Figure 4a. It can be seen that the samples CB3, CL3, and CL6 form cluster, while CL8 and CL10 assemble together, CB6 and CB8 were close to each other, CL0 were close to CB0. This grouping was further confirmed by PCoA and results were shown in Figure 4b. The variance contribution of first principal components (PC1) and second principal components (PC2) of the microbial communities were 56.03% and 17.78%, respectively, and the cumulative variance contribution was 73.81%. Cluster analysis and principal coordinate analysis showed that the storage time significantly affected the microbial community structure of chicken meat.

| Biogenic amine contents of chilled chicken during storage
Biogenic amines were considered as important indicators of meat freshness and spoilage, they have low molecular weight and consist of nitrogen-containing aliphatic, aromatic, or heterocyclic compounds and normally produced due to the decarboxylation of amino acids (Baixas-Nogueras et al., 2003). In brief, amino acids were decarboxylated either by endogenous amino acid decarboxylase that existed naturally in animal or plant cells or by exogenous enzymes generated by decarboxylase-positive microorganisms under favorable environment and conditions (Fausto et al., 2016). The spoilage microorganisms often secrete amino decarboxylase and lead to accumulation of biogenic amines in meat and meat products. The biogenic amines histamine, putrescine, tyramine, tryptamine, β-phenylethylamine, and cadaverine may be formed during storage of meat or during processing of these products (Balamatsia et al., 2006).
Naturally higher levels of spermine were found in fresh chicken (Silva & Glória, 2002;Patsias et al., 2006), which were agreed with the results of this study. Many studies showed that the contents of tyramine, cadaverine, putrescine, and histamine in refrigerated chicken meat were increasing as storage duration prolongs (Ivanov et al., 2015;Balamatsia et al., 2007;Triki et al., 2018), while spermine and spermidine were decreasing during storage (Balamatsia et al., 2006). In this study, the tyramine contents increased significantly in chicken thighs after sixth day and in breasts after eighth day of storage. Furthermore, the concentrations of putrescine and cadaverine also increased significantly in chicken thighs after sixth day and in breasts after eighth day of storage. While spermine and spermidine contents did not change significantly while stored at 4°C for 0, 3, 6 and 8 days, but increased significantly after 10th day of storage, indicating that the microbial population of arginine metabolizing increased sharply after 10th day of storage. Cadaverine and putrescine have a stale and abhorrent taste (Curiel et al., 2011), therefore refrigerated chicken at 4°C from 6th day produced faint rotten sour odor.

| Microbial community structure and diversity
Meat putrefaction is an ecological phenomenon and normally caused by prevailing of a particular microbial strains called specific spoilage organism (SSO). The prevailing of specific microbial association on fresh meat mainly relies upon initial microflora and storage conditions (Zhang et al., 2015;Hovda et al., 2007).
The initially found microflora in meat products were closely related to the environment of manufacturing enterprise, equipment, personnel, operation, and kinds of meat products (Rudy et al., 2020).
The microbial community structure and dominant microorganisms of meat products were different under different storage conditions such as Aeromonas (Zhang et al., 2015), Carnobacterium (Zhang et al., 2015), Lactococcus and Lactobacillus (Jääskeläinen et al., 2016), and Enterobacteriaceae (Kokeala & Bjrkroth, 1997)   Note: Values are mean ± standard deviation of three replicates. Bacterial diversity indices (Shannon index, Simpson index) and richness indices (Chao 1 index, ACE index) within a column with no letters in common are significantly different (p < .05).
In this study, the average relative abundance and standard devi-  (Hinton et al., 2004;Liang et al., 2012), Acinetobacter (Hinton et al., 2004), Carnobacterium (Liang et al., 2012), Brochothrix (Liang et al., 2012), and Aeromonas (Hinton et al., 2004) were main spoilage microflora found in chilled chicken stored at 4°C under aerobic conditions in previous report. This research had some difference from previous studies, and the reason may be closely related to the different initial microbial community structure in freshly slaughtered chicken produced by different enterprises (Liang et al., 2012). Pseudomonas sp., an obligate aerobe, grew optimally at 30°C, but can proliferate at temperatures as low as 4°C (Fonseca et al., 2011), so it was an absolutely dominant spoilage bacteria in chilled chicken during storage at 4°C under aerobic conditions in this study, and P. fragi, P. azotoformans, and P. cichorii were the dominant spoilage bacterial species.
Pseudomonas found in meat products has developed resistance to a variety of antibiotics (Lerma et al., 2012;Lerma et al., 2014), so its growth and reproduction should be tightly controlled.
In this study, we found that Myroides, Yersinia, and Shewanella were important contributors to the spoilage-related microflora of chilled chicken during storage at 4°C under aerobic conditions.
As a psychrophilic organism, Yersinia was able to grow at 4°C (Grahek-Ogden et al., 2007). Myroides and Shewanella also have ability to thrive at low temperatures (Hau & Gralnick, 2007), therefore the relative abundance of Yersinia, Myroides, and Shewanella increased with storage time in this study, revealing that Myroides, Yersinia, and Shewanella were potentially pathogenic to poultry safety. Therefore, cold chain transport and storage could offer a potential food safety hazard and must be controlled tightly.
Alpha diversity refers to the analysis of species richness and diversity of a single sample within a particular region or ecosystem, including Chao1, ACE, and Shannon and Simpson indices (Patrick et al., 2009). In this study, OTU coverage rates of all samples were above 99.70%, indicating that sequencing data were sufficient and biological information in the samples could be reflected completely, which can be used for the analysis of the microbial community diversity (Li et al., 2016). Chao1 and ACE indices indicated the species richness in samples, while Shannon and Simpson indices reflected species diversity (Wen et al., 2016). In case of same species richness in the community, the greater the species evenness, the greater the species diversity (Caporaso et al., 2010). In this study, Chao1, ACE, and Shannon and Simpson indices showed fluctuating and decreasing trend with storage time, revealing that bacterial diversity and richness decreased of chilled chicken during storage at 4°C under aerobic condition.
putrefaciens (Wang et al. 2014), Aeromonas veronii (Wang et al. 2014), Brochothrix thermosphacta (Nowak et al. 2011), and also lactic acid producing bacteria (Bunkova et al. 2009;Lorencova et al. 2012 correlations were for Pseudomonas spp. originated from farmed rainbow trout stored in ice and putrescine (r = .98), with cadaverine (r = .82). Yersinia rohdei and Yersinia kritensenii were identified as putrescine and cadaverine producer strains (Curiel et al. 2011). Bunkova et al. (2010) reported that the 11 bacterial strains isolated from poultry skin classified into Aeromonas genus produced putrescine, and five of them also formed cadaverine. Aeromonas verpnii revealed a strong ability to produce putrescine and cadaverine. Wang et al. (2014) believed that S. putrefaciens showed significantly higher abilities to produce putrescine than those from other genera. This would support our hypothesis that the relative abundance of Aeromonas and Shewanella were positively related to putrescine contents in this study.
In this work, we proposed that the relative abundances of Myroides and Desulfovibrio showed positive relationship with cadaverine and tyramine contents.

| CON CLUS IONS
In this work, we found the tyramine was not yielded at the initial stage of storage, however, the tyramine contents increased significantly in chicken thighs after 6th day and in breasts after 8th day of storage. Furthermore, the concentrations of putrescine and cadaverine also increased significantly in chicken thighs after 6th day and in breasts after 8th day of storage. The concentration of spermine and spermidine did not significantly change during storage until 8th day, but increased significantly after 10th day. We are grateful to Prof. Hai Jing Liu and Yong Dai for technical assistance. We also thank Feng Qiu AN, Ting Wei and Song Wang for cooperation and discussion on this manuscript.